PROJECT SUMMARY/ABSTRACT The inability to isolate pure populations of specific trophoblast (TB) subtypes from heterogeneous placental samples limits mechanistic studies on primary TB in culture, or the use of transcriptomic analysis tools for TB characterization. The central challenge in non-destructive iso- lation of TB subtypes is that the specific markers uniquely identifying them are intracellular (e.g. CDX2). There- fore, commonly used methods for isolation of specific, live cell populations, such as fluorescence activated cell sorting (FACS) or magnet-activated cell sorting (MACS), cannot be employed. To address this challenge, we pro- pose a powerful protein engineering strategy for generating non-antibody affinity reagents, which will enable isolation of specific TB subtypes from heterogeneous primary placental samples. We pro- pose to isolate CDX2+ villous cytotrophoblasts (vCTBs); this will uniquely enable the characterization of primary CDX2+ vCTBs using single-cell RNA-seq analysis, and provide insight into heterogeneity of these cells in vivo. Mechanistic studies on human TB development have been limited by the lack of an in vitro model system analogous to mouse TB stem cells; the human TB stem cell has remained elusive. In this context, TB derived from human embryonic stem cells (hESCs) has emerged as a promising model system. Isolation of specific TB populations from human placentas will enable comparison of hESC-derived TB with their in vivo counterparts. Here, single-cell RNA-seq data from primary CDX2+ vCTBs will provide a bench- mark for comparing hESC-derived CDX2+ vCTBs and primary CDX2+ vCTBs. We propose to generate non-antibody binding proteins to non-destructively isolate CDX2+ vCTBs subtypes from human placentas. Accordingly, under Aim 1, we will isolate, from a combinatorial library, affinity reagents that selectively bind to CDX2+ vCTBs, and verify that these reagents can indeed be used for separation of CDX2+ vCTBs from 6-8 wk. human placentas. Under Aim 2, we will use mass spectrometry to identify protein targets bound by the affinity reagents isolated in Aim 1. Finally, under Aim 3 we will characterize CDX2+ vCTBs from 6-8 week placentas using single-cell RNA-seq. We will also compare the transcriptomes of hESC-derived CDX2+ vCTBs and placental CDX2+ vCTBs. The significance of our proposed work relates to three expected outcomes. First, our studies will gen- erate reagents to specifically isolate specific (CDX2+) vCTB sub-populations from heterogeneous mixtures, and therefore enable their characterization using single-cell RNA-seq. Second, comparative studies on vCTBs derived from hESCs and primary placental samples will enable rigorous assessment of the suitability of hESC-derived TB as faithful models of early human TB development. Finally, our strategy can be extended to isolation of other TB subtypes, thus enabling mechanistic studies in TB biology.